Intramolecular determination of primary kinetic isotope effects in hydroxylations catalyzed by cytochrome P-450

Hjelmeland, Leonard M.; Aronow, Lewis; Trudell, James R.

doi:10.1016/0006-291x(77)90758-6

Cited by 133 publications

(53 citation statements)

References 15 publications

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“…In this event, the ratio of p:a loss from C-2 of 24 can be estimated as (53-30):30 or 1:1.3, requiring an intramolecular KIE of ca. 1.3 x 4 or 5.2, well within the observed range of such effects (18,19). The above analysis is approximate, ignoring as it does the role of intermolecular KIE's, but as these are usually close to unity in cytochrome P-450 dependent hydroxylation (19,20), their contribution is generally small.…”

supporting

confidence: 53%

“…While this result alone would indicate hydroxylation with retention of configuration (the mode of cytochrome P-450 dependent hydroxylation most commonly observed (15b)), the data obtained from the P-labelled substrate, which retains 63% of the original label, show that this cannot be the case. The analysis of the data of Table 2 depends on the relationship between stereoelectronic effects (SEE) (favouring axial (P) loss or addition of groups from C-2 (17)), and the intramolecular kinetic isotope effect (KIE) favouring loss of H over D, by a factor that may be as large as 11 in cytochrome P-450 hydroxylations (18). As outlined in Scheme 1, the loss of H from C-2 to the enzyme would be exclusively from the P face if the KIE and SEE are cooperative (for 2a-deuterium labelled substrate, 25).…”

mentioning

confidence: 99%

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Microbial hydroxylation of steroids. Part 12. Hydroxylation of testosterone and related steroids by Gnomoniafructicola ATCC 11430

Holland¹,

Brown²,

Chenchaiah³

et al. 1989

Can. J. Chem.

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The fungus Gnomonia fructicola ATCC 11430 hydroxylates testosterone at C-2P in 40-60% yield. The enzyme involved has been shown to be an inducible CO sensitive, cyanide insensitive oxygenase, with a requirement for theA4-3-ketosteroid substrate functionality. The use of 2a-and 2P-deuterium labelled substrates has shown that hydrogen loss from C-2 during 2P hydroxylation is isotope dependent and non-stereospecific. This is interpreted in terms of a A',4-dienolic enzyme-bound intermediate form of the substrate. Le champignon Gnomonia fructicola ATCC 1 1430 provoque une hydroxylation en position C-2P de la testosterone, avec un rendement de 40-60%. On a dkmontrk que l'enzyme implique une oxygknase qui peut &tre induite, qui est sensible au CO et insensible au cyanure et dont le substrat doit contenir une fonctionnalitk A4-&to-3 stkro'ide. Utilisant des substrats marquks au deutkrium dans les positions 2 a et 2P, on a pu demontrer que l'hydroxylation depend de l'isotope et qu'elle n'est pas spicifique.On peut interpreter ce resultat, en suggkrant que le substrat est lie i l'enzyme par le biais d'une forme intermediaire A2.4-diCnolique.

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supporting

confidence: 53%

mentioning

confidence: 99%

Microbial hydroxylation of steroids. Part 12. Hydroxylation of testosterone and related steroids by Gnomoniafructicola ATCC 11430

Holland¹,

Brown²,

Chenchaiah³

et al. 1989

Can. J. Chem.

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“…Although the distribution of electrons in this catalytic ferryl species remains experimentally ambiguous, it is generally formulated as a peroxidase Compound I-like intermediate, with the heme iron in the Fe(IV) state and the second oxidation equivalent stored either as a porphyrin or protein radical (6). The radical rebound mechanism described above is supported by a variety of data, including the large magnitude of the intramolecular isotope effect when the abstracted hydrogen is replaced by a deuterium (3,4,7), the observation of allylic and skeletal rearrangements during hydroxylation reactions (8)(9)(10), and density function computational analyses (6). However, one line of evidence, determination of the carbon radical lifetime by radical clock substrates, has given conflicting results.…”

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confidence: 96%

Radical Intermediates in the Catalytic Oxidation of Hydrocarbons by Bacterial and Human Cytochrome P450 Enzymes

Jiang

Montellano

2005

Biochemistry

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Cytochromes P450 cam and P450 BM3 oxidize α-and β-thujone into multiple products, including 7-hydroxy-α-(or β-)thujone, 7,8-dehydro-α-(or β-)thujone, 4-hydroxy-α-(or β-)thujone, 2-hydroxy α-(or β-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 ± 0.3 × 10 10 s −1 to 12.5 ± 3 × 10 10 s −1 for trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[ 2 H]-α-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450 cam active site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of α-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450 cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate-and enzyme-dependent.The regio-and stereoselective hydroxylation of hydrocarbons by cytochrome P450 enzymes is a chemically demanding but physiologically important reaction. Hydroxylation is required, for example, for the conversion of cholesterol into a panoply of mammalian sterol hormones and of arachidonic acid into vasodilator and vasoconstrictor signaling molecules (1,2). The mechanism of carbon hydroxylation has been thought for three decades to involve hydrogen abstraction by the catalytic ferryl species followed by recombination of the resulting substrate carbon radical with the equivalent of an iron-bound hydroxyl radical (3,4). The ferryl species involved in this reaction is generated by reductive activation of molecular oxygen at the iron † This work was supported by grant GM25515 from the National Institutes of Health.

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“…In an isotope effect experiment of intramolecular design, a substrate is chosen that is susceptible to enzymatic attack at either of two symmetrically equivalent sites (Hjelmeland et al, 1977;Miwa et al, 1980;Gelb et al, 1982;Lindsay Smith et al, 1984). One site contains deuterium and the other retains its normal complement of hydrogen.…”

Section: Symmetrical Intramolecular Designmentioning

confidence: 99%

“…A second and similar mechanism is reduction of the perferryl oxene EOS D to free substrate, free enzyme, and water Sligar, 1987, 1988;Korzekwa et al, 1989). A special experimental condition that can achieve the same end is an isotope effect experiment of symmetrical intramolecular design (Hjelmeland et al, 1977;Miwa et al, 1980;Gelb et al, 1982;Lindsay Smith et al, 1984).…”

mentioning

confidence: 99%

The Use of Deuterium Isotope Effects to Probe the Active Site Properties, Mechanism of Cytochrome P450-Catalyzed Reactions, and Mechanisms of Metabolically Dependent Toxicity

Nelson¹,

Trager²

2003

Drug Metabolism and Disposition

143

121

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:Critical elements from studies that have led to our current understanding of the factors that cause the observed primary deuterium isotope effect, (k H /k D )obs, of most enzymatically mediated reactions to be much smaller than the "true" or intrinsic primary deuterium isotope effect, k H /k D , for the reaction are presented. This new understanding has provided a unique and powerful tool for probing the catalytic and active site properties of enzymes, particularly the cytochromes P450 (P450). Examples are presented that illustrate how the technique has been used to determine k H /k D , and properties such as the catalytic nature of the reactive oxenoid intermediate, prochiral selectivity, the chemical and enzymatic mechanisms of cytochrome P450-catalyzed reactions, and the relative active site size of different P450 isoforms. Examples are also presented of how deuterium isotope effects have been used to probe mechanisms of the formation of reactive metabolites that can cause toxic effects.Historically, the determination of deuterium isotope effects has been a powerful tool to help unravel the intricacies of carbon-hydrogen bond cleavage and define the mechanism of specific chemical reactions. The intrinsic primary isotope effect, k H /k D , for a reaction is the magnitude of the isotope effect on the rate constant for the bond-breaking step (C-H versus C-D) of the reaction and is related to the symmetry of the transition state for that step. The larger the isotope effect, the more symmetrical the transition state, with the theoretical limit being 9 at 37°C in the absence of tunneling effects (Bell, 1974). The chemical events leading to the transition state and subsequent formation of products are the descriptors of a chemical mechanism. It is the relationship of k H /k D to transition state that provides mechanistic insight. Thus, k H /k D is the quantity that needs to be known, and for homogenous chemical reactions, the experimentally observed deuterium isotope effect, (k H /k D )obs, where (k H /k D )obs is defined as the ratio of a kinetic parameter such as V max or V max /K m obtained from a nondeuterated substrate to a deuterated substrate, is identical to k H /k D . This is generally not true of enzymatically mediated reactions and is the reason for the much less successful application of deuterium isotope effects to such reactions until the system was better understood. The experimentally observed isotope effect, (k H /k D )obs, was invariably found to be much smaller than k H /k D , the intrinsic isotope effect for that reaction, thereby obscuring both meaning and mechanism. Even more perplexing was the observation that (k H /k D

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Intramolecular determination of primary kinetic isotope effects in hydroxylations catalyzed by cytochrome P-450

Cited by 133 publications

References 15 publications

Microbial hydroxylation of steroids. Part 12. Hydroxylation of testosterone and related steroids by Gnomoniafructicola ATCC 11430

Microbial hydroxylation of steroids. Part 12. Hydroxylation of testosterone and related steroids by Gnomoniafructicola ATCC 11430

Radical Intermediates in the Catalytic Oxidation of Hydrocarbons by Bacterial and Human Cytochrome P450 Enzymes

The Use of Deuterium Isotope Effects to Probe the Active Site Properties, Mechanism of Cytochrome P450-Catalyzed Reactions, and Mechanisms of Metabolically Dependent Toxicity

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